All - We are trying to express for structural studies a 257 AA eukaryotic intracellular (also possibly nuclear) protein (predicted to be single domain all-helical) that has 12 Cysteines. No known metal-binding function not that it couldn't happen. So far (E. coli) it expressed solubly as MBP fusion (with an N-terminal region deleted predicted disordered) until cleavage of MBP, then it's not soluble, including detergents added. THe MBP fusion is usually soluble aggregate so we assume that our part is not folded right. We have so far assumed it needs a lot of reducing agent (5 mM DTT or TCEP). Thinking of trying chaperones and insect cells next.
Any experience out there that might help? Mostly I wonder about all the cysteines. Don't really know if that is the problem. Laurie Betts
